1. Field of the Invention
The invention relates generally to micro-arrays for contacting small quantities of chemical species. More specifically, the invention relates to micro-arrays for contacting an oligonucleotide probe with an oligonucleotide target, a reader for reading the micro-array, and a method and apparatus for making the micro-array.
2. Description of Related Art
Presently, micro-arrays are being used for a wide range of applications such as gene discovery, disease diagnosis, drug discovery (pharmacogenomics) and toxicological research (toxicogenomics). A micro-array is an orderly arrangement of immobilized chemical compounds. Micro-arrays provide a medium for matching known and unknown DNA samples based on base-pairing rules. The typical method involves contacting an array of immobilized chemical compounds with a target of interest to identify those compounds in the array that bind to the target. Arrays are generally described as macro-arrays or micro-arrays, the difference being the size of the sample spots. Macro-arrays contain sample spot sizes of about 300 microns or larger whereas micro-arrays are typically less than 200 microns in diameter and typically contain thousands of spots.
DNA micro-arrays, or DNA (gene) chips are typically fabricated by high-speed robotics on glass or nylon substrates, for which probes with known identity are used to determine complementary binding. A “probe” is a tethered nucleic acid with known sequence, whereas a “target” is the free nucleic acid sample whose identity is being detected.
One array-based application that requires very high density miniaturized arrays is sequencing by hybridization (SBH). In one common format of SBH (format II), a spatially-addressable array of the complete set of oligonucleotide probes of length n is constructed. The oligonucleotide probes are typically covalently attached to a flat, solid substrate, such as a glass slide. Each address in the array has a unique n-mer attached thereto, and the sequence of the probe is defined by its spatial address (xy coordinates). The array is contacted with a labeled target nucleic acid under conditions which discriminate between the formation of perfectly complementary probe-target hybrids and hybrids containing mismatches. Thus, only those addresses of the array which have attached thereto oligonucleotide probes that are completely complementary to a portion of the target nucleic acid produce a signal. The array is then scanned for signals, the sequences of complementary probes determined from their spatial addresses, and the sequence of the target nucleic acid determined by overlapping the common sequences of the probes.
Two other SBH formats also exist. In format I SBH, the target nucleic acid is immobilized on a solid support, e.g., a nylon or nitrocellulose filter, and the immobilized target interrogated with labeled probes. Typically, the target is interrogated with a single probe at a time, or alternatively with a plurality of probes, each of which bears a different distinguishable label (this latter mode is termed “multiplexing”). To reduce the number of manipulations required, the target nucleic acid can be spotted onto a filter in a grid or array, and each spot or address in the array interrogated with a single probe or plurality of multiplexed probes.
In yet another format of SBH, (format III), an array of immobilized oligonucleotide probes similar to that used for format II SBH is contacted with an unlabeled target nucleic acid under conditions which discriminate between perfectly complementary and mismatched hybrids. The array is then contacted with a labeled probe under conditions which discriminate between perfectly complementary and mismatched labeled probe target complexes. Following hybridization, the array is subjected to conditions which covalently join probes which are hybridized adjacently to the target (e.g., a ligase). The unligated labeled probe, and optionally target nucleic acid, is then washed away. The array is then scanned for signal. Since the solution-phase probe was labeled, only those addresses where [ligatia] ligation took place produce a signal. The sequence of the target nucleic acid is determined by overlapping the common sequences of the ligated probes.
For a review of the three types of SBH and their respective advantages, see U.S. Pat. No. 5,202,231; U.S. Pat. No. 5,525,464; WO 98/31836; WO 96/17957 and the references cited therein.
The length of target nucleic acid which can be sequenced using SBH techniques depends on the lengths of the oligonucleotide probes. Generally, sequencing a target nucleic acid a few hundred nucleotides in length requires the oligonucleotide probes to be at least 8 nucleotides in length. Sequencing longer target nucleic acids, or sequencing though regions of tandem repeats, requires even longer probes. Some have estimated that sequencing a target nucleic acid over one thousand nucleotides in length would require oligonucleotide probes of at least 12 to 14 nucleotides in length. Because the methods require the use of complete sets of probes, i.e., every possible sequence of length n, the probe sets required for the method are extremely large. For example, the complete set of 8-mer probes consists of 48 or 65,356 unique sequences. The complete set of 10-mer probes consists of 410 or 1,048,576 unique sequences and the complete set of 14-mer probes consists of 414 or 268,435,456 unique sequences. In order to make the assays practical, the entire probe array must typically be on the order of 1 cm2 in area.
To meet the needs of applications requiring high-density miniaturized arrays of immobilized compounds, such as SBH and its related applications, two general methods have been developed for synthesizing the immobilized arrays: in situ methods in which each compound in the array is synthesized directly on the surface of the substrate and deposition methods in which pre-synthesized compounds capable of being covalently attached to the surface of the substrate are deposited, typically by way of robot dispensing devices, at the appropriate spatial addresses. The in situ methods typically require specialized reagents and complex masking strategies, and the deposition methods typically require precise robotic delivery of very defined quantities of reagents.
For example, Fodor et al., 1991, Science 251:767-773 describe an in situ method which utilizes photo-protected amino acids and photo lithographic masking strategies to synthesize miniaturized, spatially-addressable arrays of peptides. This in situ method has recently been expanded to the synthesis of miniaturized arrays of oligonucleotides (U.S. Pat. No. 5,744,305). Another in situ synthesis method for making spatially-addressable arrays of immobilized oligonucleotides is described by Southern, 1992, Genomics 13:1008-1017; see also Southern & Maskos, 1993, Nucl. Acids Res. 21:4663-4669; Southern & Maskos, 1992, Nucl. Acids Res. 20:1679-1684; Southern & Maskos, 1992, Nucl. Acids Res. 20:1675-1678. In this method, conventional oligonucleotide synthesis reagents are dispensed onto physically masked glass slides to create the array of immobilized oligonucleotides.
U.S. Pat. No. 5,807,522 describes a deposition method for making micro arrays of biological samples that involves dispensing a known volume of reagent at each address of the array by tapping a capillary dispenser on the substrate under conditions effective to draw a defined volume of liquid onto the substrate.
One of the biggest drawbacks of both the in situ and deposition micro fabrication techniques is the inability to verify the integrity of the array once it has been fabricated. Absent analyzing the compound immobilized at each address, the integrity of the deposition chemistry simply cannot be verified. Such an analysis would be extremely labor intensive, and may even be impossible for extremely high-density arrays, as the quantity of compound immobilized may not be sufficient for analysis and subsequent use.
Moreover, since each array is fabricated de novo, the integrity of each array synthesized is suspect. Without being able to verify that the array has been fabricated with high fidelity, the absence of a signal at a particular address cannot be unambiguously interpreted. The absence of signal could be due to a failed synthesis or immobilization at that address.
Deposition methods suffer additional drawbacks, as well. Automatic deposition generally uses a robotic fluid delivery system. The robot moves to specific locations on the microcard, delivering a specified amount of fluid. The fluid is deposited onto the microcard by either a non-contact ejector (such as ink jet nozzles) or a contact ejector (such as a pen, quill, or fiber) which actually touches the microcard surface to release the fluid. Ink jets, pens, and quills are adaptations of common devices, and each have reliability problems. Ink jets work fine when the fluid has been carefully optimized for the nozzle. However, when depositing many different fluids through the same nozzle, optimization of each fluid is impractical. Pens and quills are very useful for deposition onto a small number of plates but are too slow for cost-effective production. While a fiber piston delivery system shows promise as a reliable means of fluid deposition, it requires an unwieldy number of fibers for a very large number of reaction sites.
In addition to problems with the reliability of ejectors, the total time to deposit thousands of different probe fluids with existing automated ejector devices increases the cost of a microcard beyond the cost of other approaches, i.e. the automated process is not cost-effective. Somewhat surprisingly, this is not due to the speed of fluid deposition by the robot, which is relatively fast. Rather, it is the combination of other on-line procedures such as wicking, cleaning, and loading slides that makes the total deposition time unacceptable. To have thousands of independent probe liquids means that the robot can only deposit a few spots on one slide (assuming some duplication) before it has to load (wick) another probe fluid into the reservoirs of its ejector or quill. Wicking usually involves providing an open vessel containing the probe fluid such that the robot can move the ejector/quill into the fluid and load the fluid through vacuum or capillary action into a reservoir in the ejector/quill. This process can take several seconds, and must be conducted whenever dispensing a new probe fluid. Also, before introducing a new probe fluid, the ejector/quill must be cleaned to prevent contamination of the new probe fluid with the previous one. This cleaning usually involves flushing the ejector/quill with a cleaning solvent and drying them with flowing gas. The cleaning process also takes several seconds and must be conducted whenever dispensing a new probe fluid. Furthermore, the loading (and unloading) of slides into the robot's workspace also adds to the overall processing time. Since wicking, cleaning, and loading are on-line procedures, they all add to the total time of deposition. Spotting many slides at a time improves the robotic deposition time but still requires the same wicking and cleaning time before depositing a different fluid. Therefore, wicking, cleaning, and loading time alone make the process too time consuming and expensive to consider as a viable alternative.
Consequently, neither in situ nor existing automated approaches are a reliable or cost-effective means of mass-producing micro arrays. Therefore, there is a need for methods of making microarrays that avoid the problems associated with currently available in situ and deposition methods and which provide a matrix or array of contact points for contacting small quantities of at least two chemical species. Furthermore, there is a need for a more advantageous structure for the micro arrays that can provide an increased number of mix points as well as an improved contact efficiency between the chemical species.
Machines for synthesizing chemical chains or compounds onto a solid substrate have been in existence for many years. Typically such synthesizers make oligonucleotides by adding one phosphoramodite (base) at a time onto solid beads. The bases, A, T, C or G, are strung together into a chain of the desired sequence and length. The process of adding these bases may vary from manufacturer to manufacturer. The solid substrate is usually a batch of small polystyrene or glass beads (typically less than 1 mm diameter). A plurality of beads are placed in a container and fluids are passed through the beads. The process usually comprises adding these bases by the following process (1) detritylation, (2) applying base A, T, C or G, (3) adding an activator, (4) applying caping agent A and B, (5) washing with a first solvent, (6) applying an oxidizer, and (7) washing with a second solvent. This process adds one base onto the beads and is repeated for each base desired. The only process variable is the base A, T, C or G which is determined by the compound or chain desired. After all the desired bases are added, the oligos are cleaved (separated) from the beads by an ammonia solution. An extraction process, such as High-pressure Liquid Cromotography (HPLC), separates and purifies the oligos from the ammonia. The final oligo product is in a liquid form that is often marked and stored before being used or sold. The user, typically using a robot, must then conduct another set of steps to deposit and immobilize the liquid oligos onto a solid substrate for analysis purposes.
The disadvantage with existing synthesizers is that the final product is often not application ready. The product is in a liquid form that must typically be inventoried, stored, and usually reapplied onto another substrate, such as a titer-plate or micro-slide, to be analyzed. A more efficient process would be to synthesize the oligos on the same substrate that is ultimately analyzed. Furthermore, if the synthesis process could be automated such that the substrate is continuously fed through the solution, rather than the solution being fed through the substrate, the synthesized product could be placed directly onto the analysis device without the need for inventory, storage, or re-application.